We report the detection of hypervariable markers in the satyrid butterfly Bicyclus anynana (Butler) using the human-derived minisatellite probes 33.6 and 33.15. A highly informative fingerprinting system was established, and, uniquely to date, probes 33.6 and 33.15 were found to hybridize to an identical single hypervariable locus at low stringency in this species. Segregation analysis was performed using two families, consisting of two parents and 15 and 16 offspring, respectively. Mendelian inheritance was confirmed. The number of alleles present and the level of heterozygosity were determined by fingerprinting 18 unrelated individuals: 11 alleles were observed, and 83% of individuals were heterozygous. These data show for the first time that hypervariable markers occur in the genome of a butterfly species; they suggest that additional loci may be revealed by hybridization with other minisatellite probes. Both probes detected one other independently segregating polymorphic locus to which 33.15 hybridized more intensely, allowing genotypes to be characterized. It is possible that similar methods could yield useful results in other Lepidopteran species.